Glioblastoma multiforme (GBM) and breast cancer are major health burdens that will benefit from new mechanistic insights that lead to novel management strategies. We propose to investigate Signal Transducer and Activator of Transcription (STAT)3, thioredoxin reductase (TrxR)1 cytoplasmic isoform 3, and glucose-6-phosphate 1-dehydrogenase (G6PD) isoform a as the targeting mechanisms underlying the antitumor responses of GBM and triple-negative breast cancer to hirsutinolide compounds. The hirsutinolide series are the major chemical constituents of Vernonia cinerea. This plant has traditionally been used in Asia to treat specific ailments and in clinical trials proofed to be effective to moderate type 2 diabetes and curtail smoking. Despite the therapeutic potential of the hirsutinolides, however, little was known of their pharmacological properties to facilitate their clinical development until our studies. Our compelling data show that structurally suitable hirsutinolides strongly inhibit STAT3, TrxR1 and G6PD functions and thereby suppress breast cancer and GBM phenotypes in vitro and in vivo. Initial structure activity relationship analysis showed a critical requirement for a position 13 ester group for the activities. Unbiased computational modeling/docking and nuclear magnetic resonance structural studies show that structurally suitable hirsutinolides bind the STAT3 DNA-binding domain (DBD) via hydrophobic interactions between the position 13 side chain and specific amino acid residues in the STAT3 DBD. Select compounds caused mitotic and cell cycle arrests and inhibited growth of human breast cancer and GBM xenografts. In line with the TrxR1 or G6PD functional roles in the cellular protection against the oxidative damage from excessive reactive oxygen species (ROS) or in the pentose phosphate pathway for NADPH production, respectively, ROS levels and glycolytic metabolites, including glucose, G6P and pyruvate are significantly altered in hirsutinolide-treated tumor cells. We hypothesize that structurally suitable hirsutinolides are inherently active against STAT3, TrxR1 and G6PD functions, and alter cellular redox events and glycolytic metabolism, and thereby block breast cancer and GBM progression. We will determine the SAR of the hirsutinolides relative to inhibition of STAT3 activity and elucidate the structural determinants for STAT3 inhibition (Aim 1), investigate the mechanistic details of the STAT3 signaling inhibition by hirsutinolides and the significance to the antitumor responses against GBM and breast cancer (Aim 2), define the mechanistic link between STAT3, TrxR1 and G6PD functions, altered cellular redox recycle, metabolism, mitosis and cell cycle (Aim 3), and study the antitumor efficacy responses of select hirsutinolide analogs and their correlation to STAT3, TrxR1 and G6PD-dependent molecular and metabolomic events in vivo (Aim 4). Data will ultimately facilitate the development of the hirsutinolides as potential therapeutics for breast cancer and GBM.